Flores M, Aristoy M C, Toldrá F
Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain.
Biochimie. 1993;75(10):861-7. doi: 10.1016/0300-9084(93)90040-y.
An aminopeptidase B from porcine skeletal muscle was successfully purified by ammonium sulphate fractionation and HPLC anion-exchange. The purified aminopeptidase B eluted at 0.18 M NaCl, had a relative molecular mass of 76,000 Da and was markedly stimulated in the presence of 0.2 M chloride anion. The enzyme exhibited maximum activity for the hydrolysis of the arginine-aminoacyl bond at pH 6.5 and 37 degrees C. Other substrates consisting of phenylalanine, proline and alanine-aminoacyl bonds were cleaved at 5.9, 5.1 and 2.5% of the maximum activity with the arginine-aminoacyl bond. The enzyme did not show endopeptidase activity and was very stable at pH above 6 and temperatures below 35 degrees C. However, the enzyme inactivated very fast when incubated at pH 5 or at 50-65 degrees C. Bestatin (50 microM) completely inhibited the aminopeptidase B activity while EDTA (5 mM) only inhibited 40% of its activity. However, 0.5 mM of E-64 did not cause any inhibition while 0.05 mM amastatin and 1 mM puromycin only inhibited 11% of the enzyme activity.
通过硫酸铵分级分离和高效液相色谱阴离子交换法成功纯化了猪骨骼肌中的氨肽酶B。纯化后的氨肽酶B在0.18 M氯化钠条件下洗脱，相对分子质量为76,000 Da，在0.2 M氯离子存在时受到显著刺激。该酶在pH 6.5和37℃时对精氨酸 - 氨酰键的水解表现出最大活性。由苯丙氨酸、脯氨酸和丙氨酸 - 氨酰键组成的其他底物的水解活性分别为精氨酸 - 氨酰键最大活性的5.9%、5.1%和2.5%。该酶不显示内肽酶活性，在pH高于6和温度低于35℃时非常稳定。然而，在pH 5或50 - 65℃孵育时，该酶失活非常快。贝司他汀（50 microM）完全抑制氨肽酶B的活性，而乙二胺四乙酸（5 mM）仅抑制其40%的活性。然而，0.5 mM的E - 64没有引起任何抑制作用，而0.05 mM的氨抑素和1 mM的嘌呤霉素仅抑制该酶11%的活性。
